An overall objective is to complete and extend studies initiated with the support of a previous PHS grant (AM 11223) which dealt with the mechanism of allosteric regulation of carbamyl phosphate synthetase (CPSase) from E. coli. The propose studies involve obtaining information from several different approaches which will complement understanding of the catalytic and regulatory mechanism(s) of carbamyl phosphate carbamyl phosphate synthesis in other systems (eukaryots) and to other enzymes whose mechanisms are related to the mechanism of CPSase. The studies which are proposed are based on previous and current investigations and include 1) study of the binding of allosteric effectors (IMP, UMP, ornithine) by equilibrium dialysis, 2) determination of whether the enzyme is catalytically active as a monomer and whether the regulatory properties of the enzyme are manifested when the enzyme exists as a monomer (immobilizied CPSase, gel filtration chromatography, and density gradient centrifugation procedures will be employed), 3) development of affinity chromatography procedures for isolating CPSase from different sources, 4) Kinetic studies, inluding initial velocity, product inhibition, analog inhibition (phosphonate analogs of carbonate- phosphate anhydride and carbamyl phosphate), isotope exchange kinetic studies of the partial reactions catalyzed by CPSase, and 5) utilization of cyanate as an analog of glutamine to investigate interactions between the small and large subunits of CPSase as it is related to the regulatory properties of the enzyme.